Treatment and inhibition of disease conditions using flexible heteroarotinoids

ABSTRACT

The present invention contemplates methods of treating, reducing, inhibiting or preventing several diseases by the administration of flexible heteroarotinoids. Among the diseases or conditions which can benefit from treatment with flexible heteroarotinoids as described herein are, (1) cancers and other diseases that involve abnormal differentiation, (2) diabetes, (3) hemophelia, (4) liver disease, (5) diseases involving human aldehyde dehydrogenase 2, (6) polycystic kidney disease, (7) lysosomal storage diseases, (8) high cholesterol, (9) obesity, (10) high triglycerides, (11) glycoprotein metabolism diseases, and (12) diseases involving abnormal angiogenesis.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 11/404,701,filed Apr. 16, 2006, now U.S. Pat. No. 7,612,107, which claims thebenefit under 35 U.S.C. 119(e) of U.S. Ser. No. 60/671,692, filed Apr.15, 2005, the entirety of each of which is hereby expressly incorporatedherein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

BACKGROUND OF THE INVENTION

The majority of cancer-related deaths occur after primary therapy hasbeen completed, mostly due to recurrence of the cancer or development ofsecond cancers. Major efforts are underway to develop pharmaceuticalsthat can prolong the disease-free interval after primary therapy bypreventing recurrence of the cancer or the development of new cancers.Only agents that lack significant toxicity are acceptable in thissetting. One of the most promising classes of cancer chemopreventionagents designated by the Chemoprevention Working Group to the AmericanAssociation for Cancer Research (AACR) is retinoids (1). Thesecompounds, which are modeled after the active vitamin A metabolite,retinoic acid (RA), offer promise as cancer chemoprevention agentsbecause of their abilities to regulate growth, differentiation,apoptosis, angiogenesis, metastasis and immune function. Despite limitedsuccess of various isomers of RA (All-trans-RA, 13-cis-RA and 9-cis-RA)and a synthetic retinoid in chemoprevention trials (Fenretinide, 4-HPR),structural alterations of the compounds are needed to improve thetherapeutic ratio (efficacy/toxicity) before clinical application of aretinoid strategy for chemoprevention (2-5).

The toxicities associated with chronic retinoid treatment affect theskin, mucus membranes, hair, eyes, gastrointestinal system, liver,neuromuscular system, endocrine system, kidneys and bone, and arecollectively termed hypervitaminosis A (6). These individual toxicitiesand teratogenicities have been shown to be induced through activation ofnuclear retinoic acid receptors (RARalpha, RARbeta, RARgamma) andretinoid X receptors (RXRalpha, RXRbeta, RXRgamma) that act astranscription factors (7, 8). Early efforts to improve the therapeuticratio involved constraining the RA double-bonds, by inclusion in anaromatic ring of chemical structures called arotinoids. The firstarotinoid evaluated, TTNPB, was 10-fold more potent than RA inbiological assays of efficacy, but considerably more toxic (9-12).

Our strategy to reduce the toxicity of arotinoids was to retardmetabolic oxidation of the compounds by incorporation of oxygen orsulfur heteroatoms to replace one of the gem-dimethyl groups in thetetrahydronaphthalene ring of TTNPB (FIGS. 1A-1B). The resultingcompounds, called Heteroarotinoids (Hets), exhibited the similarbiological activities to RA (9, 13), but significantly reducedtoxicities (12). Thus, inclusion of the heteroatom in the arotinoidstructure was shown to greatly improve the therapeutic ratio(efficacy/toxicity) in animal models (9, 12). The clinical applicationof a Het called Tazarotene (produced by Allergan) for treatment ofpsoriasis, has confirmed the improved therapeutic ratio predicted forcompounds with heteroatoms (14).

Individual structural alterations of Hets greatly affected theirselectivities for individual RAR and RXRs (FIG. 1) (12, 15-17). A Hetthat activated RXRs only (OHet72) was found to be sufficient to inhibitestablishment head and neck xenograft tumors, while a retinoid thatactivated both RARs and RXRs (SHet50) exerted greater growth inhibitoryactivity (17). The importance of RARgamma activation in skin cancer wasdemonstrated by comparisons of Hets, which differed by single structuralalterations that regulated their abilities to activate the RARgreceptor. A Het that activates all six nuclear receptors (NHet90)induced significantly greater growth inhibition of vulvar carcinoma celllines in comparison to structurally related compounds, that activate allretinoid receptors except RARgamma (NHet17 and NHet86) (16).Interestingly, Hets containing three-atom urea or thiourea linkers,which increased the flexibility of their conformations, regulated growthand differentiation similar to RA, but did not activate the RARs andRXRs (18). These flexible Hets (Flex-Hets) exhibited significantlygreater growth inhibition activity against epithelial ovarian cancer andborderline-cancer cells than benign epithelial ovarian cells and normalendometrial cells (19). The most potent Flex-Het, SHetA2, wascharacterized for the mechanism of this strong growth inhibition in headand neck cancer cell lines, and was found to induce apoptosis through G2cell cycle arrest, alterations in mitochondrial membrane permeability,release of cytochrome c from the mitochondria, generation of reactiveoxygen species (ROS), and activation of caspase 3 (19, 20). Generationof ROS was also demonstrated in SHetA2-treated ovarian cancer cell lines(19).

While natural RA isomers and classical retinoids are weak apoptosisinducers, some retinoids, 4-HPR, CD437/AHPN and MS3350-1, which areselective for RARgamma activation, exhibit potent apoptosis-inducingactivity similar to Flex-Hets (21). 4-HPR also weakly activates RARbeta,and activation of multiple retinoid receptors by 4-HPR is involved inthe mechanism of growth inhibition in leukemia cells (22, 23). Theadditional non-retinoid activities possessed by these compounds have ledto their classification as retinoid-related molecules (RRMs). While theability of these compounds to induce apoptosis is only partiallyindependent of the retinoid receptors, Flex-Hets are unique in that theyinduce apoptosis completely independent of RAR and RXR activation (20,24, 25). Several clinical trials of 4-HPR demonstrated limited cancerchemoprevention activity at low doses, and tolerable toxicity at higherdoses sufficient to induce apoptosis (3, 5, 26).

Identification of therapies for inhibiting angiogenesis is an importantarea of research. A multitude of experiments have demonstrated that thedevelopment of blood vessels is required to support tumor growth andmetastasis. Studies of human tumor specimens found that the number ofvessels within tumors correlates with disease stage and patientprognosis. The levels of circulating angiogenic cytokines also correlatewith prognosis in cancer patients. The migration of endothelial cells toform blood vessels within tumors is a complex process involving cancercells, growth factors, fibroblasts, extracellular matrix turnover.Physical contact between endothelial cells and fibroblasts is requiredfor the differentiation of endothelial cells into tubes with branches.

Strategies for therapeutic angiosuppression generally involve eitherinterference with the activators of angiogenesis or amplification of theendogenous suppressors. The classes of angiogenesis antagonists incurrent clinical trials include inhibitors of proteases, endothelialcell migration and proliferation, angiogenic growth factors, matrixproteins on the endothelial cell surface, such as integrins, copperantagonists, and other inhibitors with unique mechanisms.

For example angiogenic inhibitors in current clinical trials include (1)protease inhibitors such as marimastat, BAY 12-9566, Af 3340, andNeovastat, (2) inhibitors of endothelial cell migration andproliferation such as TNP-470, squalamine, combretastatins, endostatin,angiostatin, penicillamine, (3) antagonists of angiogenic growth factorssuch as anti-VEGF antibody, thalidomide, sugen-5416, antiangiogenicribozyme, SU 6668, interpheron-alpha and suramin, (4) inhibitors ofendothelial-specific Integrin/Survival signaling such as Integrinantagonists (e.g. Vitaxin), (5) copper antagonists/chelators such aspenicillamine, tetrathiomolybdate and captopril, and (6) angiogenicinhibitors with distinct mechanisms such as ABT-627, CM101,Interleukin-12, IM862 and PNU145156E.

Other areas of research involve therapies for treating lysosomal storagediseases and polycystic kidney disease.

Several approaches to develop experimental model systems for the studyof endometrium have been attempted. The earliest studies of endometriumwere performed using animal models such as the Rhesus monkeys. In humanstudies, organ cultures cut from hysterectomy specimens have been usedas a model system, but were limited by variability between specimens andinability to cycle the endometrium in vitro. Cultured endometrial cellscollected from hysterectomy specimens, peritoneal fluid or curretage ofthe endometrium have also been utilized after separating the stromalcells from the epithelial glands. More recently, cells collected frommenstrual secretions have been successfully grown in tissue culture.Although these studies have been successful at culturing normalendometrial cells in monolayers, the systems used are highly artificialand do not mimic the in vivo architecture or intracellular communicationof the human endometrium. Three dimensional organotypic cultures ofendometrial cells isolated from surgical specimens and grown in collagenI or basement membrane material (Matrigel) have been used in attempts todevelop experimental systems for the study of human endometrium. Thesecultures demonstrated characteristics of endometrial architecture butwere limited by only occasional formation of gland-like structures andconsiderable shrinkage of the collagen gels over prolonged treatmenttimes.

In addition to steroid hormones, retinoic acid, the natural metaboliteof vitamin A, is involved in the maintenance and regulation ofdifferentiation in the cycling endometrium and the decidualization offibroblasts. Throughout the menstrual cycle, the intracellular levels ofretinoic acid and expression of cellular retinoid binding proteinsfluctuate, while nuclear retinoic acid receptors remain at similarlevels. Epidemiological studies have found that high dietary intake ofvitamin A and carotenoids have been associated with a decreased risk forendometrial and ovarian cancer. Retinoic acid was shown to delay tumorinduction in hamster cheek pouch epithelium exposed to 25 μg DMBA. Thepapillary epidermoid carcinomas that developed were less invasive andless keratinized in the retinoic acid treated animals than in theanimals treated with DMBA alone.

Autosomal dominant polycystic kidney disease is one of the most commoninherited disorders in humans, occurring in 1 in 500 to 1 in 1000individuals. The cost of this disease is enormous as it is the thirdleading cause of end-stage renal disease. Costs are further escalated byextra-renal complications such as hypertension, cystic liver disease andintracranial aneurysms. Therefore, any therapy that can slow theprogression of this disease would be of great benefit.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows chemical structures of parent retinoid compounds and Hets(MTD=Maximum Tolerated Dose).

FIG. 1B shows chemical structures of representative Flex-Hets used inthe present invention.

FIG. 2 shows graphs of in vitro growth inhibition of the National CancerInstitute's Human Tumor Cell Line Panel. Each line represents thedose-response growth inhibition curve of the individual cell lines asindicated in the graph legend (CNS=central nervous system).

FIG. 3 shows Flex-Het induction of Mitochondrial Swelling. Ovariancancer cells (A2780) were treated with the Flex-Hets SHetA2, SHetA3 andSHetA4 or the same volume of solvent for the indicated amount of time.The JC1 dye was then used to measure mitochondrial swelling. The lightcurve represents untreated cultures and the dark curve representscultures treated with SHetA2. An increase in fluorescent (Fl.) intensity(rightward shift) represents mitochondrial swelling. The bottom twographs show the reduced effects of SHetA2 on normal ovarian surfaceepithelial cells and normal endometrial cells.

FIG. 4 shows graphs demonstrating inhibition of xenograft tumor growthby Hets and 4-HPR. Groups of 5 mice bearing OVCAR-3 xenografted tumorswere gavaged daily with the indicated drugs in sesame oil (▪ closedsquare) or with sesame oil alone (∘ open circle). Tumor volumes weremeasured in 3 dimensions with calipers weekly. Each data pointrepresents the average and standard error of the tumor volumes on theindicated treatment day relative to the tumor volume on day zero. Nu/numice were injected subcutaneously with one million OVCAR-3 cells andarbitrarily divided into five groups of six mice each. After three weeksof tumor growth, the animals were gavaged with sesame oil, 10 mg/kg/day4-HPR, or the indicated Het five times per week for four weeks and thetumors were measured once per week. The tumor sizes were averaged anddivided by the average size for each group on the day that treatment wasinitiated (day 0).

FIG. 5 shows effects of Flex-Hets and 4-HPR on gland formation, MUC-1expression and apoptosis in tumors of treated animals. Statisticallysignificant higher levels of glands and MUC-1 expression in the tumorsof treated animals in comparison to controls treated with sesame oil areindicated by asterisks (*) and were determined by t-tests. For glandformation, p<0.001 for SHetA2, p=0.097 for SHetA4, p=0.001 for SHet50,and p=0.007 for 4-HPR. For MUC-1 scores, p=0.011 for SHetA2, p=0.050 forSHetA4, p=0.040 for SHet50, and p=0.001 for 4-HPR. The higher levels ofTUNEL scores in the tumors of treated animals in comparison to controlanimals approached significance (t-test, p=0.27 for SHetA2, p=0.48 forSHetA4, p=0.20 for SHet50, and p=0.12 for 4-HPR).

FIG. 6 shows graphs demonstrating the association of topical irritancywith RAR/RXR activation. Female Skh hairless mice, 6-8 weeks old, weretreated topically on the dorsal skin for 4 days over a 2-logconcentration dose range with various compounds. Retinoic acidreceptor-Hets were evaluated in A, while retinoic acidreceptor-independent Flex-Nets were evaluated in B. Daily flaking andabrasion scores were combined to calculate a single cutaneous toxicityscore for each mouse. Each treatment group consisted of 4 mice, andgroup averages were used to plot cutaneous toxicity against doses of thecompounds.

FIG. 7 shows micrographs of EAhy.963 endothelial cells were plated onmatrigel in a multi-well plate and various concentrations of SHetA2 wereadded to the wells in triplicate. Twenty hours later an investigator whodid not know the treatments administered to each well scored the numberof branches in each well and took representative photomicrographs. Thegraph on the left provides quantification of the endothelial branchesfor each treatment dose. The graph on the right demonstrates that thegrowth of EAhy.926 endothelial cells is less sensitive to the effects ofSHetA2 than the A2780 and OVCAR-3 ovarian cancer cell lines.

FIG. 8. Effect of 5 μM SHetA2 on blood vessel density in the CAM assay.The mean±SEM blood vessel density per treatment as compared tobFGF/VEGF-treated CAMs; *=p<0.05 and **=p<0.001 as compared tobFGF/VEGF-treated CAMs as determined by one-way ANOVA and Neuman-Keulspost-test. These data are representative of two separate experimentswith an n=7-22.

FIG. 9. SHetA2 inhibition of Angiogenesis. C6 glioma cells were injectedinto the brains of 2 rats. One week later, one rat was gavaged with 60mg/kg/day SHetA2 on each Monday, Wednesday and Friday. Blood vesselswere imaged by MRI.

FIG. 10. SHetA2 inhibition of blood flow. C6 glioma cells were injectedinto the brains of 2 rats. One week later, one rat was gavaged with 60mg/kg/day SHetA2 on each Monday, Wednesday and Friday. The blood flowwas measured by MRI imaging.

FIG. 11. SHetA2 inhibition of glioma tumor growth. C6 glioma cells wereinjected into the brains of 2 rats. One week later, one rat was gavagedwith 60 mg/kg/day SHetA2 on each Monday, Wednesday and Friday. The tumorsize was measured by MRI imaging.

FIG. 12. Various Xenograft tumors stained with CD34 antibodies (brown)to identify endothelium. The B16 mouse melanoma cell line wasestablished as a syngeneic tumor in an immunocompetent mouse model andthe human Caki renal cancer and A2780 ovarian xenograft tumors inimmunocompromised mice. The treated mice were gavaged with 60 mg/kg/daySHetA2 and the untreated were gavaged with solvent only. The tumors wereexcised and sections were stained with the anti-CD34 antibody to detectendothelial cells (brown stain).

FIG. 13. Real Time rt-PCR validation of SHetA2 inhibition of TP andTSP-1 mRNA Expression. OVCAR-3 cultures were treated with 1 μM SHetA2for the indicated period of time prior to harvesting the cells andisolating RNA. cDNA was prepared and used in real time rt-PCR reactions.Real time rt-PCR was performed on cDNA from cultures various treatmenttimes. Similar results were observed for A2780 cultures.

FIG. 14. Western blot validation of SHetA2 inhibition of TP and TSP4protein expression. A2780 ovarian cancer cells were treated with 10 μMSHetA2 for the indicated amounts of time. Equal amounts of whole cellprotein extracts were evaluated by Western blot.

FIG. 15. SHetA2 Inhibition of NF-κB. A2780 cultures were transfectedwith the NF-κB reporter and treated with 10 μM SHetA2 for the indicatedtimes. Cell lysates were prepared and evaluated for luciferase activitywhich was normalized for protein concentration.

FIG. 16 shows a graph related to apoptosis. SW962 vulvar carcinomacultures were treated with 10 μM of the indicated retinoid or the samevolume of solvent. At the specified times, the cultures were labeledwith Annexin-V-Fluos and propidium iodide (PI), and the number ofapoptotic cells was quantified using dual-laser flow cytometricanalysis.

FIG. 17. Retinoid effects on ovarian carcinoma organotypic cultures.Organotypic cultures of OVCAR-3 (A, B, E, F, I and J), Caov-3 (C and G),SK-OV-3 (D and H), and O2 (K and L) were grown in the absence (−, toprow and K) or presence (+, middle row and I, J and L) of retinoid HetA4at a concentration of 1 μM in the media. All sections were stained withH&E except for I, which was stained with PAS to detect glycoproteins andglandular differentiation, and J, which was stained with the TUNEL assayto detect apoptosis. All magnifications are 40×.

FIG. 18 shows graphs demonstrating that Thymidine Phosphorylase InhibitsSHetA2-Induced Apoptosis. The A2780 ovarian cancer cell line was treatedwith 10 FM SHetA2 for 0, 20 and 22 hours in the presence and absence ofthymidine phosphorylase. Apoptosis was measured with Annexin-V-FITC andFlow Cytometry.

FIG. 19 shows micrographs showing untreated cells, cells treated withDMBA carcinogen only, SHetA2 only, or DMBA+SHetA2. Untreated: Thespecimen is cellular with both surface and mid-gel cells presence inabundance. Two cell types are clearly present. One appears to bestromal. The nuclei are thin and spindle shaped and the cytoplasm issimilar. When cut in cross-section the nuclei are very small and roundand hyperchromatic. The other cells have large, oval to almost boxcarnuclei with small nucleoli and abundant cytoplasm. Numerous mitosis arepresent. The epithelial cells are almost syncitial in nature and appearto cling to one another. With DMBA Carcinogen: The specimen is cellular.The cells present have lost their cytoplasm. There is more pleomorphismof the cells and many are very hyperchromatic. Mitoses are verynumerous-approximately 2× the number in untreated. Cells no longer aretogether but are distinct one from another. Stromal cells intermix withthe epithelial cells. With SHetA2 Chemoprevention Agent: The specimen iscellular but the overall cellularity appears to be increased incomparison due to less apoptosis and decreased in comparison to DMBAheated because of increased apoptosis. Mitosis are still increased aboveuntreated but less than DMBA treated. The cells more closely resembleuntreated. In comparison to the DMBA treated, they have more cytoplasmand are clearly less pleomorphic. With DMBA and SHetA2: The specimen ismarkedly reduced but still abundant cells remain. The cause isapoptosis. Mitosis are still present but the least number of all 4treatments. There is so much apoptosis that the cells do not clingtogether. Overall the cells remaining have cytoplasm, very littlepleomorphism and resemble untreated. Both cell types are present. Alinear discriminant function which utilized five texture features,including total optical density and nuclear area, was developed todistinguish between untransformed and DMBA-transformed nuclei of thefirst cell line (epithelial cells only). The resulting vector of featureweights was then applied to the karyometric data for nuclei from cellstreated with both DMBA and SHet2A, as well as untransformed cellstreated with SHet2A alone. The value of each feature in the treatedgroups was standardized relative to the untreated control. Thus, thestandardized measure for each feature in each nucleus in the tissueunder study indicates how may normal-tissue standard deviations awayfrom the normal-tissue value each nucleus lies.

FIG. 20. Mice harboring Caki renal cancer xenografts were treated with60 mg/kg/day SHetA2 or PEG400 vehicle for 15 days. After another 15 daysof growth, the tumors were removed, fixed, embedded and sections.Sections were stained with PAS to identify glycoproteins and lysosomes.The untreated tumor is on the left and the treated tumor is on theright.

FIG. 21. Effects of Flex-Hets on formation of cysts in culturedpolycystic kidney disease cells. A primary culture of polycystic kidneycells were grown inside a mixture of collagen I and matrigel and treatedwith prostaglandin E2 to induce cyst formation. Parallel cultures weretreated with 10 micromolar SHetA3 and SHetA4.

DESCRIPTION OF THE INVENTION

The present invention contemplates methods of treating and inhibitingseveral diseases by the administration of flexible heteroarotinoids(Flex-Hets), including for example SHetA2, SHetA3, SHetA4, and SHetC2(FIG. 1B). Our Oklahoma-based retinoid research group has developed aseries of low-toxicity retinoids, flexible heteroarotinoids, which havedemonstrated significant activity against cancer tissue and abnormallydifferentiated tissue from multiple tissue types. In another embodimentflexible heteroarotinoids are used to inhibit angiogenesis and treatsubjects having polycystic kidney disease (PKD) and lysosomal storagediseases (LSDs) as discussed in more detail below.

Flexible heteroarotinoids contemplated for use in the present inventioninclude, but are not limited to, those described below and as shown inU.S. Pat. No. 6,586,460 which is hereby expressly incorporated byreference herein in its entirety.

Examples of flexible heteroarotinoids which can be used in the presentinvention include, but are not limited to, compounds having the isomericformula:

in which:

G denotes H or CH₃;

R denotes H, CH₃, or OCH₃;

Q denotes H or i-C₃H₇;

W denotes O or S; and

Z denotes NO₂, CO₂Et, CO₂-n—C₄H₉ or SO₂NH₂;

and compounds having the isomeric formula:

in which:

G denotes H or CH₃;

R denotes H, CH₃, or OCH₃;

Q denotes H or i-C₃H₇;

W denotes O or S; and

Z denotes NO₂, CO₂Et, CO₂-n-C₄H₉ or SO₂NH₂;

and compounds having the isomeric formula:

in which:

G denotes H, CH₃, C(O)—CH₃;

R denotes H, CH₃, or OCH₃;

W denotes O or S;

Q denotes H or i-C₃H₇; and

Z denotes NO₂, CO₂Et or CO₂-n-C₄H₉.

Flexible heteroarotinoids are compounds that regulate growth,differentiation and apoptosis, but do not directly activate retinoicacid receptors (RARs) or retinoid X receptors (RXRs). SHetA2 and SHetA4,for example (as shown herein), inhibited the growth of ovarian cancerxenografts, renal cancer xenografts, and glima othotopic tumors withoutevidence of toxicity and has improved survival in mouse models ofmelanoma and ovarian cancer. Additional animal models have demonstratedthat SHetA2 does not induce teratogenicity or skin irritation. Thus,SHetA2 exhibits an improved therapeutic ratio over retinoids capable ofactivating the retinoid receptors.

Without wishing to be constrained by theory, it is hypothesized thatSHetA2 (and other Flex-Hets) directly interacts with the mitochondriacausing mitochondrial swelling within 30 minutes of treatment andresulting in activation of the intrinsic apoptosis pathway. This directaction is independent of generation of reactive oxygen species (ROS) andregulation of mRNA and protein synthesis because antioxidants (to quenchROS), Actinomycin D (to prevent mRNA synthesis) and cyclohexamide (toprevent protein synthesis) do not prevent mitochondrial swelling andapoptosis induced by SHetA2, SHetA3, and SHetA4. Mitochondria in cancercells are more susceptible to this activity than normal cells becausecancer cells have greater rates of metabolism and mitochondrialmutations than normal cells. The more stable mitochondrial state ofnormal cells makes them more resistant to SHetA2-induced apoptosis.

In one embodiment of the invention, the flexible heteroarotinoids, e.g.,SHetA2, appear to regulate gene expression by binding to and/or actingon hepatocyte nuclear factor-4 (HNF-4), Nuclear Factor kappa B (NfκB)and serum response factor (SRF) and other immediate early transcriptionfactors and nuclear receptors, which have specific DNA binding sitespresent in the promotors of genes regulated by SHetA2.

In another embodiment of the invention, SHetA2 regulation of expressionof the thymidine phosphorylase, thrombospondin-4 and other genes whichregulate the development of blood vessels. The ultimate effect ofregulating these genes is to prevent the development of blood vesselswithin tumors (antiogenesis), thus starving the tumor of oxygen andother nutrients needed for growth.

In one embodiment, the Flex-Hets of the present invention areadministered to subjects exposed to a carcinogen thereby preventingdevelopment of the abnormal cancerous phenotype (abnormaldifferentiation) and inducing apoptosis in cells that are otherwise ableto survive and become cancerous.

One objective of the present work was to demonstrate the value ofFlex-Hets as anti-cancer pharmaceutical agents. Positive controlsincluded RA isomers (all-trans-RA, 9-cis-RA), pan-agonist Hets thatactivate all RARs and RXRs (SHet50, NHet17), an RRM that activatesretinoid receptors (4-HPR) and/or an agonist Het selective for RARgamma(SHet65) (15) (See FIGS. 1A and 1B for exemplary structures). Otherflexible heteroarotinoids contemplated for use herein are describedabove, and in U.S. Pat. No. 6,586,460. The ranges of growth inhibitoryactivities were evaluated in 59 cell lines representing 10 cancer types.In vivo activity and toxicity was evaluated in an ovarian cancerxenograft animal model and in animal models of skin irritation andteratogenicity.

Materials and Methods

Drugs

The Hets were synthesized and their receptor specificity determined aspreviously described (15-17 and U.S. Pat. No. 6,586,460). 9-cis-RA waspurchased from Biomol and 4-HPR was provided by Johnson and Johnson.Drugs were dissolved differently for each in vitro and in vivo assay asdescribed below.

In Vitro Cytotoxicity Assays in Cervical Carcinoma Cell Lines.

The SHetA2, SHetA3, SHetA4, SHet50 and 9-cis-RA compounds were evaluatedin 4 cervical carcinoma cell lines. The SiHa, CC-1 and C33a humancervical cell lines were maintained in Minimal Essential Media (MEM)containing Earle's salts and L-glutamine supplemented with nonessentialamino acids, 1% sodium pyruvate, 10% fetal bovine serum (FBS) andantibiotic/antimycotic. The HT-3 cervical carcinoma cell line wascultured in McCoy's 5a medium supplemented with 10% FBS andantibiotic/antimycotic. Cells were inoculated into 96 well microtiterplates at densities of 1000 cells/well and incubated for 24 h prior toaddition of drugs. Drugs were dissolved in dimethyl sulfoxide (DMSO) anddiluted in complete medium, prior to addition to triplicate culturewells to achieve final concentrations of 1, 4, 7 and 10 mM. Followingdrug addition, the plates were incubated for 72 h. The assay wasterminated by the addition of cold TCA and the cells were stained with0.4% sulforhodamine B (SRB) in 1% acetic acid. Bound stain wassubsequently solubilized with 10 mM trizma base, and the absorbance wasread on an automated plate reader at a wavelength of 560 nm. Growthinhibition was determined by dividing the average Optical Density (OD)of the triplicated treated cultures by the average OD of the triplicatecontrol cultures treated with DMSO solvent only. This ratio wasconverted into a percentage by multiplying by 100. The potency(concentration required to induce half of the maximal activity—GI₅₀)were derived from dose-response graphs generated with results from 3 to5 individual experiments using GraphPad Software.

SHetA2 Cytotoxicity in NCI Human Tumor Cell Line Panel

The SHetA2 compound was evaluated in the National Cancer Institute (NCI)human tumor cell line panel by the Developmental Therapeutics Program(DTP). The human tumor cell lines were grown in RPMI 1640 mediumcontaining 5% fetal bovine serum and 2 mM L-glutamine. Cells wereinoculated into 96 well microtiter plates at densities ranging from5,000 to 40,000 cells/well depending on the doubling time of individualcell lines and incubated for 24 h prior to addition of SHetA2. After 24h, two plates of each cell line were fixed in situ with TCA, torepresent a measurement of the cell population for each cell line at thetime of drug addition (Tz). Drugs were dissolved in dimethyl sulfoxide(DMSO) and diluted in complete medium containing 50 μg/ml gentamicin,prior to addition to the culture wells. Final drug concentrations rangedfrom 10⁻⁴ to 10⁻⁸ M in a series of 10-fold dilutions. After 24 h, twoplates of each cell line were fixed in situ with TCA, to represent ameasurement of the cell population for each cell line at the time ofdrug addition (Tz). Following drug addition, the plates were incubatedfor an additional 48 h and then the assay was terminated by the additionof cold TCA and stained as described in the preceding section. Theabsorbance was read on an automated plate reader at a wavelength of 515nm. Using the seven absorbance measurements (time zero, (Tz), controlgrowth, (C), and test growth in the presence of drug at the fiveconcentration levels (Ti)), the GI₅₀ was calculated from[(Ti−Tz)/(C−Tz)]×100=50. The NCI results are presented as the averageand standard deviation of two independent experiments.

Xenograft Tumor Animal Model.

The OVCAR-3 cultures were maintained in RPMI media supplemented with 10%fetal bovine serum. All animal experimentation described in thismanuscript was conducted in accord with accepted standards of humaneanimal care. Twenty-five female NU/NU CD-1 female mice (Charles RiversLaboratories) were housed in a laminar flow room under sterileconditions at 83-85° F. The mice were quarantined for one week prior tothe beginning of the study and were allowed access to autoclaved food(Purina 5001 mouse/rat sterilizable diet, St. Louis, Mo.) and water adlibitum. OVCAR-3 cells in log phase growth were harvested bytrypsinization, resuspended in RPMI culture medium, and centrifuged at3,000 rpm for 10 min. The pellets were resuspended in RPMI culturemedium at a concentration of 7×10⁶ cells/ml before implantation intomice. Animals were injected with 3.5×10⁶ cells into the right scapularregion with a 24-gauge needle/1 cc tuberculin syringe. Twenty-four hoursafter tumor implantation, animals were randomized into 5 groups of 5animals each.

The Hets SHet50, SHetA2 and SHetA4 were synthesized and their receptorspecificity determined as previously described (17, 19). The 4-HPRcompound was a gift from R.W. Johnson Pharmaceutical Research Institute,Raritan, N.J. All retinoids were dissolved in super refined sesame oil(Croda, Inc., Parsippany, N.J.) and stored at −80° C. Since theretinoids are light sensitive, all manipulations involving retinoidswere performed under subdued lighting. Drugs were administered daily bygavage beginning 35 days after tumor implantation with a 20-gaugeintragastric feeding tube (Popper & Sons, New Hyde Park, N.Y.), 5days/week, at 10 mg/kg/day in 0.1 ml of oil for each treatment group. Acontrol group received 0.1 ml of oil without retinoid. Tumors weremeasured with calipers weekly, and tumor volumes were calculated usingthe formula: volume=length×width×height. Tumor growth was determined bythe dividing the volume of the tumor at each weekly measurement by thevolume on the first day of treatment. Animal weights and clinical signsof overall health status and cutaneous toxicities were recorded weekly.

Histochemical and Immunohistochemical Evaluation of Xenograft Tumors

On the last day of treatment, the animals were sacrificed and then thetumors were removed, fixed in neutral buffered formalin and embedded inparaffin. Five micron sections of the paraffin-embedded tumors werestained with hematoxylin and eosin (H&E) for histologic evaluation, andwith the TUNEL (terminal deoxynucleotidyl transferase-mediatedbiotin-deoxyuridine triphosphate nick) FragEL kit (Oncogene ResearchProducts, Boston, Mass.) for evaluation of apoptosis according tomanufacturers instructions. MUC-1 expression was detected in tumorsections using a mouse monoclonal antibody to human MUC-1 (Pharmingen,San Diego, Calif.) and the Histostain-Plus AEC Kit (Zymed Laboratories,San Francisco, Calif.) according to manufacturers instructions. Briefly,endogenous peroxidase activity in deparaffinized sections was quenchedwith 3% H₂O₂ in methanol and then sections were incubated in serumblocking solution, and subsequently in primary antibody, diluted 1:50 inphosphate buffered saline (PBS), overnight at 4° C. in a humid chamber.Sections were then washed with PBS, treated with biotinylated secondaryantibody for 15 minutes, followed by enzyme conjugate for 10 minutes.AEC (3-amino-9-ethyl carbazole) chromagen was applied for 15 minutesfollowed by counterstaining with Harris' hematoxylin. Sections of amucinous gastric carcinoma were used as a positive control (New cornerSupply, Middleton, Wis.). Omission of the primary antibody was used as anegative control. Both the MUC-1 and the TUNEL stains were repeatedthree times on separate sections cut at three different levels of thetumors. The slides were coded so that laboratory personnel and thepathologist were blinded from the treatment groups. Tumor sections werescored for number of glands (quantified in one H&E stained sections and3 MUC-1-stained sections) and MUC-1 and TUNEL staining (based onstaining intensity and percent positive cells). Scores from threeseparate sections taken from different locations within the tumors wereaveraged. A student's t-test was used to compare the scores between thedifferent treatment groups. P values of less than 0.05 were consideredstatistically significant.

Alanine Aminotransferase (ALT) Activity

At the end of the treatment period, blood was drawn from each animal.Plasma was separated by centrifugation and stored at −70° C. Theactivity of ALT was determined in aliquots of plasma by standardspectrophotometric enzymatic techniques which are based on the reductionof pyruvate by lactate dehydrogenase (28). The method measures NADHdisappearance with time of incubation at 37° C. The activities areexpressed as mm/L/min. Two different amounts of plasma were used in eachassay to assure measurement of the maximum rate.

Topical Irritancy

Topical irritancy was evaluated according to published procedures (29).Briefly, female Skh hairless mice, 6-8 weeks old, were treated topicallyon the dorsal skin for 4 days over a 2-log concentration dose range withvarious compounds. Daily flaking and abrasion scores were combined tocalculate a single cutaneous toxicity score for each mouse. Eachtreatment group consists of 4 mice, and group averages are used to plotcutaneous toxicity against dose of the retinoid compound.

Generation and Analysis of Retinoid-Rescued Embryos.

Generation of Raldh2_/_ embryos (from matings of heterozygous parents)and treatment with retinoids and heteroarotinoids were performed asdescribed for all-trans-RA, which eliminates the block in growth at E8.5observed in untreated mutants (44). Briefly, retinoids were dissolved incorn oil and administered orally to timed-pregnant Raldh2_/_ mice at12-h intervals on E7.25, E7.75, and E8.25. Dosages varied from 0.025 to25 mg/kg. At E10.25, embryos were analyzed morphologically to determinewhether Raldh2_/_ embryos were rescued or whether wild-type embryos hadsuffered teratogenesis. Detection of embryonic RA was performed in situin embryos carrying the RARE-lacZ RA-reporter gene by staining forbeta-galactosidase activity for 10 h (45).

Quantitation of RA in Embryos, Placentas, and Serum.

All-trans-RA and 9-cis-RA were quantitated by HPLC analysis of embryos,placentas, and maternal serum from untreated or retinoidtreated pregnantmice. For pregnant mice treated by oral gavage, all-trans-RA or 9-cis-RAwere dissolved in corn oil, a single dose was administered on E10.5ranging from 2.5 to 50 mg/kg, and tissues were collected 2 h afteradministration (or 1 h where indicated). For dietary RA treatment,all-trans-RA was dissolved in corn oil and mixed with powdered food at0.1 mg/g of food for treatment on E7.5 and at 0.25 mg/g of food fortreatment on E8.5-E10.5 as described (46), and tissues were collected atE10.5. For each wild-type pregnant mouse examined, three samples wereprepared at stage E10.5: (i) all embryos were pooled (˜8-10); (ii) allplacentas were pooled (˜8-10); and (iii) 0.2 ml of maternal serum wascollected. All extraction and analytical procedures were carried out ina darkened room to protect the retinoids from exposure to light. Serum(0.2 ml) was mixed with 0.2 ml of 0.25 M ammonium acetate (pH 4.0) and0.6 ml of acetonitrile. Embryo and placenta samples (0.1-0.2 g) weremixed with 0.3 ml of 0.25 Mammonium acetate (pH 4.0) and 0.6 ml ofacetonitrile and homogenized on ice. After centrifugation (10,000˜g for10 min at 4° C.) the supernatant was transferred to a new tube, and 0.4ml of water was added. The resulting mixture was loaded onto a precolumn(Pelliguard LC-18; Supelco) and then into the analytical column asfollows. Reversed-phase HPLC analysis was performed on a Waters 2695HPLC system by using a SUPLEX pkB-100 analytical column (250×4.6 mm)(Supelco) at a flow rate of 1 ml/min and column temperature of 35° C.Mobile phase consisted of 2% ammonium acetate/glacial aceticacid/acetonitrile/methanol (16:3:79:2). Detection of retinoids wasperformed by using a photodiode array detector (Waters model 2996),which collected spectra between 200 and 450 nM. Standard solutions ofretinoids (all-trans-retinol, 9-cis-RA, and all-trans-RA) were used toobtain the calibration curves. Characteristic peak spectra and retentiontimes were used to identify each retinoid, and peak areas at A max usedfor quantitation were calculated by using MILLENNIUM CHROMATOGRAPHYMANAGER software (Waters).

Quantitation of RA in Adult Liver.

Mouse liver (1.0 g) was homogenized in 2 ml of PBS (0.01 M, pH 7.4), andthen 3 ml of methanol was added and mixed by vortex. This mixture wasextracted twice with 5 ml of hexane. Hexane layers were collected,combined, and evaporated under vacuum. The residue was dissolved in 0.15ml of mobile phase, and 0.1 ml was analyzed by using the HPLC/photodiodearray detector as described above (without precolumn) to identify andquantitate all-trans-RA and 9-cis-RA.

Results

In Vitro Cytotoxicity of Multiple Cell Lines.

Three structurally-related Flex-Nets, SHetA2, SHetA3 and SHetA4, wereevaluated for their ability to inhibit the growth of cervical cancercell lines over a range of concentrations. Since these compounds do notactivate the RARs and RXRs they were compared to the most potent RAR/RXRpan-agonist Het (SHet50) and RAR/RXR pan-agonist RA isomer (9-cis-RA).Each compound inhibited growth of all cell lines in the micromolarrange. The efficacies of the compounds were compared by measuring themaximal growth inhibition, defined by the percentage growth inhibitioninduced by 10 mM drug in comparison to the untreated control (Table 1).The Flex-Hets exhibited statistically significant greater efficaciesthan the receptor-active compounds across all cell lines as determinedby a two-tailed paired t-test (p<0.05). The efficacies of the receptoractive Het (SHet50) on the other hand, were not significantly differentthan 9-cis-RA across all cell lines (two tailed paired t-test: p>0.05).The potencies were compared by deriving the GI₅₀ values (concentrationsthat induced half of the maximal growth inhibition activity) from graphsof growth inhibition versus drug concentration (summarized in Table 1).SHetA2 consistently exhibited the greatest efficacies and potencies incomparison to all other compounds tested against the cervical cancercell lines, as was observed in previous studies for ovarian cancer celllines (19) and head and neck cancer cell lines (20). The cervical cancercell lines however, were more resistant to Flex-Nets, than ovarian andhead and neck cancer cell lines, which exhibited greater than 90% growthinhibition when treated with 10 μM Flex-Hets in previous studies (19,20).

TABLE 1 Cell Line SHetA2 SHetA3 SHetA4 SHet50 9-cis-RA SiHa 68% 58% 67%35% 24% 3.8 ± 2.9 mM 7.5 ± 0.2 mM 5.9 ± 2.3 mM 11.9 ± 1.2 mM 5.5 ± 0.7mM p = 0.038 p = 0.047 p = 0.013 p = 0.173 CC-1 58% 42% 59% 15% 28% 2.3± 0.1 mM 7.1 ± 0.3 mM 6.5 ± 0.1 mM ND ND p = 0.002 p = 0.047 p = .013 P= 0.143 C33a 87% 84% 85% 67% 45% 3.3 ± 0.6 mM 4.8 ± 1.3 mM 4.3 ± 0.8 mM3.9 ± 0.1 mM 5.6 ± 0.6 mM p = 0.023 p = 0.005 p = 0.025 p = 0.189 HT-392% 65% 76% 47% 47% 3.9 ± 0.1 mM 7.2 ± 1.7 mM 5.9 ± 0.9 mM 5.5 ± 0.1 mM8.6 ± 1.1 mM p = 0.049 p = 0.030 p = 0.042 p = 1 The efficacy is thepercent growth inhibition relative to the untreated control induced by10 mM compound. The potency is the concentration required to induce halfmaximal activity (GI₅₀) ND = not determined. The p values are from atwo-tailed paired t-test comparing the efficacy of the Flex-Hets orSHet50 versus 9-cis-RA. Efficacy (top line), Potency (middle line) andStatistical Significance (bottom line) of Flex-Hets (SHetA2, SHetA3 andSHetA4) and a Receptor Active Het (SHet50) versus 9-cis-RA on CervicalCancer Cell Lines.

To evaluate the spectrum of cancers sensitive to Flex-Hets, the mostpotent compound, SHetA2, was submitted to the National Cancer Institutes(NCI's) human tumor cell line screen for evaluation in 55 cell linesrepresenting 9 different cancer types. All cell lines representingleukemia, non-small cell lung cancer, colon cancer, central nervoussystem cancer, melanoma, ovarian cancer, renal cancer, prostate cancerand breast cancer were growth inhibited with GI₅₀ values in themicromolar range (FIG. 2).

Flex-Hets Target the Mitochondria

Swelling of mitochondrial in cancer cells was observed within 30 minutesof treatment with Flex-Nets SHetA2, SHetA3, and ShetA4, and thisswelling was maintained for the 24 hours leading up to cell death (FIG.3). This swelling was not prevented by co-treatment with inhibitors ofRNA or protein synthesis or by antioxidant inhibitors of reactive oxygenspecies generation indicating that the mitochondrial effects are theresult of direct action of Flex-Nets on mitochondria. SHetA2significantly regulated expression of 7 mitochondrial genes in ovariancancer cells (Table 2).

TABLE 2 Gene Name Genbank ID Fold ATP-binding cassette, sub-family B(MDRTAP), member 6 (ABCB6), NM_005689.1 0.46 nuclear gene encodingmitochondrial protein Translocase of inner mitochondrial membrane 44(yeast) homolog NM_006351.1 0.50 (TIM44) Sulfite oxidase (SUOX), nucleargene encoding mitochondrial protein NM_000456.1 2.11 A kinase anchorprotein S-AKAP84 mRNA, nuclear gene encoding U34074.1 2.14 mitochondrialprotein A kinase (PRKA) anchor protein 1, clone MGC: 1807 BC000729.12.00 aarF domain containing kinase 4 BC013114 2.30 3-hydroxyisobutyratedehydrogenase, mitochondrial precursor AK025558 0.00 Flex-Het RegulatedMitochondrial Genes. Genes that were expressed at statisticallysignificant higher (>1 Fold) or lower (<1 Fold) levels in ovarian cancercells treated with Flex-Het 11 (SHetA2) were identified by microarrayanalysis.

In Vivo Inhibition of Xenograft Tumor Growth.

The in vivo efficacies of the two strongest Flex-Hets, SHetA2 andSHetA4, and the strongest RAR/RXR pan-agonist Het, SHet50, wereevaluated in an animal xenograft model using the OVCAR-3 ovarian cancercell line. The well-characterized RRM, 4-HPR, was administered forcomparison, and sesame oil only served as a negative control. Drugtreatment was initiated after the tumors were established and growing,which was 35 days after injection of the tumor cells into the animals.Since the ultimate route of administration in humans will be oral, thedrugs were administered by gavage 5 days per week for 4 weeks. At theend of the treatment, the xenograft tumors were extremely heterogeneousin size ranging from 36 to 2200 mm³, which is reflective of theheterogeneous population of cells characteristic of the OVCAR-3 cellline. Each of the compounds significantly inhibited the growth of thexenograft tumors (FIG. 4). After 15 days of treatment, the growth of thetumors in each treatment group were significantly less than the growthof the control group treated with sesame oil alone (t-tests on day 29:p=0.028 for 4-HPR; p=0.010 for SHet50; p=0.046 for SHetA2; P=0.033 forSHetA4). There were no significant differences between the degrees ofgrowth inhibition exerted by the different compounds.

Differentiation and Apoptosis in Xenograft Tumors.

At the end of the experiment, the xenograft tumors were evaluated forhistology, differentiation and apoptosis. Differentiation in ovariancancer is routinely defined by gland formation and mucin expression.Pathologic review revealed that the tumors were arranged into largeirregular nests and islands of cells separated by thin strands offibrous connective tissue (FIG. 5). The majority of epithelial cellnests exhibited a differentiated phenotype, however smaller areas ofdedifferentiated cells also were observed within the tumors. In thedifferentiated areas, there were small glands and gland-like structures,with some of the cells lined up in a duct-like pattern. Occasionallypapillations were present. The majority of cells in these differentiatedareas were quite large with abundant cytoplasm and pleomorphic nucleicontaining clumped chromatin and prominent nucleoli. In thededifferentiated areas, the sizes of cells were markedly reduced, thecytoplasm was not apparent, and the nuclei tended to be more spindle inshape and were very hyperchromatic.

The tumors from the treated animals exhibited more differentiatedcharacteristics in comparison to the untreated control tumors. As seenin FIG. 5, cells in the treated tumors are arranged in a flatter moreorganized fashion instead of being piled up one cell on top of the otheras in the untreated culture. Even more clearly the “punched out” holesin the treated cultures are glands that have formed. The numbers ofglands in 3 sections taken from different areas of each tumor werequantified and compared between the different treatment groups. Tumorsfrom each of the three treatment groups exhibited significantly greaternumbers of glands than the untreated control group (FIG. 5). Thereceptor-independent Het, SHetA2, exhibited the greatest degree of glandinduction.

To evaluate the induction of differentiation at the molecular level, 3sections taken from different areas of each tumor were stainedimmunohistochemically for expression of the mucin-1 (MUC-1) protein.MUC-1 expression was noted in the differentiated areas of the tumorswith specific expression in the apical surface of glandular lumens, butnot in the areas of dedifferentiated cells (FIG. 5). An experiencedPathologist evaluated each section for the percentage of positivelystained cells and the intensity of staining and provided a score thatincorporated both parameters. The average scores of the sections foreach treatment group were compared. All drugs significantly increasedthe level of MUC-1 expression in the tumors (FIG. 5). Apoptosis wasmeasured in tumor sections by the TUNEL assay (FIG. 5). Although thelevels of apoptosis appeared higher in the treated tumors, the increasewas not statistically significant.

Evaluation of Oral Toxicity.

During the course of the xenograft mouse model experiment, the animalswere monitored daily for visually-observable signs of retinoid toxicityto the skin or bone. None of the retinoids induced evidence of the skinor bone toxicities, which were observed in previous experiments whenanimals were treated with 10 mg/kg/day of the all-trans RA isomer (17).To determine if 4-HPR and the Hets induced hepatotoxicity in this animalmodel, plasma alanine aminotransferase (ALT) activity levels weremeasured and histological liver sections were examined at the end of thetreatment period. There were no statistically significant differencesbetween the ALT activities in the different treatment groups or betweenthe treatment groups and the control group (Table 3, t-test: p>0.05).Each of the ALT values was in the normal range of ALT values (28-184mM/L/min) for this species of mouse as reported by the supplier (CharlesRivers Laboratories, Wilmington, Mass.). Necrosis, fatty changes orinflammation in portal tracts were not observed in histologic evaluationof liver sections.

TABLE 3 ALT Activity Treatment (mm/liter/min) Sesame Oil Control 103.0 ±60.3  4-HPR 149.7 ± 140.9 SHet50 65.5 ± 46.3 SHetA2 118.5 ± 153.4 SHetA364.5 ± 40.3 *Normal range 18-184 (mM/L/min). ALT Activity in Plasma ofMice

Skin Irritancy

The potential for utilizing Hets for treatment of cervical, vulvar,melanoma and other skin cancers as a topical formulation will dependupon the levels of irritancy induced by the compounds. Therefore,topical irritancy of receptor-agonist Hets and Flex-Hets were evaluatedin an animal model in comparison to all-trans-RA. A pan-agonist Het thatactivates all RARs and RXRs (NHet17) and a Het selective for RARgamma(SHet65) induced topical irritancy scores in a dose-responsive mannersimilar to all-trans-RA (FIG. 6A). In contrast, the RXR-specificretinoid OHet72 (FIG. 6A), and the RAF/RXR-independent Flex-Hets,SHetA2, SHetA3 and SHetA4 (FIG. 6B), did not induce topical irritancy inthis model.

The results described above demonstrate that Flex-Hets exhibit improvedtherapeutic ratios over other Hets, as well as natural and syntheticretinoids, which are RAR and/or RXR agonists. Despite the lack of RAR orRXR activation by Flex-Hets at the molecular level, these compoundsexhibit retinoid-like activities such as growth inhibition anddifferentiation induction at the cellular level. On the efficacy side ofthe therapeutic ratio, the growth inhibition induced by Flex-Nets isgreater than the RAR/RXR-active compounds in cervical cancer cell lines(Table 1). Previous studies in ovarian and in head and neck cancer celllines demonstrated that this high level of growth inhibition is due toinduction of apoptosis (18-20). Apoptosis is likely to contribute to themechanism of growth inhibition induced in leukemia, non-small cell lung,colon, central nervous system, melanoma, renal, prostate and breastcancer as indicated by the negative percent growth levels induced by 10and 100 mM SHetA2 in the majority of cell lines of the NCI's human tumorcell line panel (FIG. 2). Although 10 mM SHetA2 concentrations arerequired to induce apoptosis in a 48 hour treatment period, our previousstudy demonstrated that longer treatment times with 1 mM SHetA2, SHetA3,SHetA4 and 4-HPR could induce apoptosis in a more biologically relevantorganotypic culture model of ovarian cancer (18). Interestingly, all ofthe other potent apoptosis-inducing RRM's characterized to date, 4-HPR,CD437/AHPN and MS3350-1, are selective for RARgamma (21), suggestingthat the RRM's and possibly Flex-Hets induce apoptosis through anunidentified nuclear receptor with a similar ligand binding pocket tothat of RARgamma.

In contrast to the in vitro results from OVCAR-3 organotypic cultures,induction of apoptosis was not observed in vivo in the OVCAR-3 xenografttumors in this study. The lack of apoptosing cells measured at the endof the treatment period however, does not preclude the possibility thatapoptosis could have contributed to the growth inhibition observedduring the treatment period. In the in vivo situation, cells inducedinto apoptosis by the treatments could have been eliminated through theinnate immune system present in these animals (30). In the in vitroassays however, there is no biological system to eliminate apoptoticcellular debris, and therefore sufficient numbers of cells couldaccumulate to allow detection of drug-induced apoptosis (18). The trendtoward higher percentages of apoptosis in the tumors from all treatmentgroups in this study however, indicates that apoptosis did contribute tothe mechanism of growth inhibition by these drugs in vivo.

The lack of evidence for oral toxicity or topical irritancy induced byFlex-Nets in this study is consistent with their lack of RAR/RXRactivation. The lack of teratogenicity induced by SHetA2 in a mousemodel is also consistent with RAR/RXR independence (31). An additionaltoxicity that needs to be avoided in improving retinoids is thepotential harm that can be done to patients who continue to smoke duringtreatment. Clinical trials for prevention of second cancers have foundthat RA and β carotene, which is a nutrient that can be metabolized toRA, may be harmful to patients if they continue smoking during treatment(32-34). Although the exact mechanism of these harmful effects is notclear, these findings demonstrate the importance of developing novelpharmaceuticals, such as Flex-Hets, that can induce the same anticanceractivities as retinoids, but through different molecular mechanisms.

A mouse model that lacks retinaldehyde dehydrogenase (Raldh2) due tonull mutations was used to test the ability of Flex-Hets to substitutefor retinoic acid (RA) and induce birth defects. Null mutations ofRaldh2 have demonstrated that RA is essential for mouse development,because Raldh2_/_ embryos lack mesodermal RA (both all-trans-RA and9-cis-RA) and fail to develop beyond embryonic day (E)8.5 (43, 44). Weexamined the relative ability of all-trans-RA, 9-cis-RA,receptor-specific synthetic retinoids and retinoic acid receptorindependent Flex-Hets to rescue the Raldh2_/_ embryonic lethalphenotype.

Treatment of the pregnant mothers with 0.5 mg/kg RAR-specific SHet100also resulted in the typical rescue phenotype observed with all-trans-RAplus the same RARE-lacZ expression phenotype). Treatment with 0.25 mg/kgSHet100 resulted in a partial rescue of Raldh2_/_ embryos, similar tothat observed with 2.5 mg/kg 9-cis-RA, and 2.5 mg/kg SHet100 wasteratogenic for all embryos in a litter (Table 4). Treatment with 2.5mg/kg of either RXRspecific OHet72 or non-receptor-binding SHetA2 (Table4) resulted in no rescue, with Raldh2_/_ embryos developing the same asuntreated embryos and having the RARE-lacZ expression pattern typical ofnonrescued mutants. Increasing the dosage of OHet72 and SHetA2 to 10 or25 mg/kg also resulted in no rescue, and no teratogenesis was observedin litters treated with OHet72 or SHetA2 at any dosage (Table 4).

Our results indicate that an RAR-specific ligand can provide the samerescue phenotype as all-trans-RA, whereas SHetA2 and other compoundsthat do not activate the RAR receptors cannot provide any degree ofrescue. These findings provide evidence that Flex-Nets cannot substitutefor RA and are not teratogenic.

TABLE 4 Rescue of E10.25 Raldh2−/− embryos by various retinoids Dose,Total no. No. or +/+ No. of −/− No. of −/− Retinoid mg/kg of embryos or−/+ unrescued rescued Teratogenesis* All-trans-RA 1.0 19 12 7 0 0All-trans-RA 2.5 41 27 1 13  0 9-cis-RA 2.5 30 20 8 2 (partial) 09-cis-RA 10 30 23 1 6 0 SHet100 0.025 11 9 2 0 0 SHet100 0.25 10 8 0 2(partial) 0 SHet100 0.5 20 13 1 6 0 SHet100 2.5 11 — — — 11 OHet72 2.5 84 4 0 0 OHet72 10 19 16 3 0 0 OHet72 25 17 12 5 0 0 SHetA2 2.5 8 7 1 0 0SHetA2 10 18 13 5 0 0 SHetA2 25 7 6 1 0 0 Embryos indicated as +/+(wild-type) or −/+ (Raldh2-heterozygous) were normal in appearance withRARE-lacZ expression throughout the trunk mesoderm, whereas −/−(Raldh2-heterozygous) unrescued embryos were much smaller and failed toundergo axial rotation, and −/− rescued embryos were normal in size butwith growth-retarded for limb buds and no RARE-lacZ expression insomites or lateral plate mesoderm. *Teratogenesis was reported aspositive after observation of an obvious growth defect or malformationin +/+ or −/+ embryos. In the single instance where teratogenesis isindicated, all embryos in that litter failed to undergo axial rotationand were stalled between stages E8.5 and E9.0.

The lead Flex-Het, SHetA2, exhibited the greatest efficacy and potenciesof all of the other Flex-Nets and retinoids tested. This greateractivity was not associated with any detectable increase in oraltoxicity, skin irritancy or teratogenicity. It could be postulated thatthe most active Flex-Het would also be the most potent, but greaterlevels of toxicity or irritancy were not observed in SHetA2-treatedanimals in comparison to animals in the other treatment groups. Thissupports the previous observation that the induction of apoptosis byFlex-Nets occurs through a mechanism that is selective for cancer cellsover normal cells (19). The efficacy, toxicity, pharmacokinetics andformulation of SHetA2 are currently being evaluated in the NationalCancer Institute's (NCI's) Rapid Access to Intervention Development(RAID) program (Application 196, Compound NSC 726189). The RAIDpharmacokinetic studies demonstrated that micromolar concentrations ofSHetA2 can be achieved and maintained in mice (35) indicating thatconcentrations sufficient to differentially induce apoptosis in cancercells over normal cells can be targeted in clinical trials.

In conclusion, Flex-Hets exhibit improved therapeutic ratios formultiple cancer types over RAR and/or RXR agonists, in that they exertsimilar effects on growth, differentiation and apoptosis withoutactivating the RARs and RXRs and without inducing the oral toxicity,skin irritancy and teratogenicity associated with receptor activation.

Inhibition of Angiogenesis

In another embodiment of the present invention, in a method ofregulating angiogenesis, the flexible heteroarotinoid is administeredand causes the regulation of expression of the thymidine phosphorylase(also called platelet derived endothelial cell growth factor, PD-ECGF)and expression of TSP-4. The Flex-Het may be administered to endothelialcells to prevent their ability to develop into blood vessels. TheFlex-Het may be administered to animals or humans to prevent thedevelopment of blood vessels in disease states such as cancer, diabetesand eye disease. Also envisioned is the use of Flex-Nets to inhibitangiogenesis of blood vessels in other conditions which are potentiallydeleterious to the host, including, but not limited to, telangiectasias,arterial malformations, venous malformations, capillary malformations,lymphatic malformations, arterio-venous malformations, hemangiomas, oraneurysms. In addition, the invention envisions using the describedproteins to treat areas characterized by the growth of structurallynormal blood vessels in a manner or site that may compromise the wellbeing of the host, sometimes collectively referred to as areas of“neovascularization”. It is envisioned that Flex-Nets would beadministered to the host (by any of the variety of routes describedherein) in order to inhibit localized angiogenesis in a therapeuticmanner.

Multiple models demonstrate that SHetA2 inhibits angiogenesis andregulates expression of genes that control angiogenesis in vitro and invivo. The in vivo models include the conventional chorioallantoicmembrane (CAM) assay, Magnetic Resonance Imaging of a Rat glioma tumor,histologic evaluation of melanoma, ovarian and renal cancer tumors inmouse models. The in vitro models include an endothelial tube formationassay and microarray analysis.

The well-documented angiogenic activity of TP and the implication ofTSP-4 in angiogenesis, both of which are inhibited by SHetA2, suggestedthat SHetA2 might inhibit angiogenesis. A dose-responsive inhibition ofangiogenesis was confirmed in an in vitro assay of endothelial tubebranching (FIG. 7). The increased thickness of the vessels at 1 μM andthe increasing proportions of solitary clumps of cells at 5 and 10 μMindicate that SHetA2 is inhibiting endothelial cell migration. Todetermine if this anti-angiogenic activity was due to growth inhibitionor cytotoxicity. The viability of the endothelial cells treated withSHetA2 for 24 hours was evaluated. The graph in FIG. 7 demonstrates thatthe EAhy.926 endothelial cells were much less growth inhibited by SHetA2than the two ovarian cancer cell lines, A2780 and OVCAR-3. This confirmsthe therapeutic ratio observed in previous studies.

Chorioallantoic Membrane (CAM) Assay.

The conventional model for demonstration of anti-angiogenic activity isthe Chorioallantoic membrane (CAM) assay. The CAM assay that was used isbased upon the original procedure by Jakob et al. (36) withmodifications made by our laboratory (37, 38) (see FIG. 8).

Rat Glioma (Brain Cancer) Model.

In this in vivo model, glioma cells are injected into the brains of ratsand the growth of the tumor and development of blood vessels in thetumor is measured by magnetic resonance imaging (MRI). FIGS. 9-11demonstrate that oral administration of 60 mg/kg/day SHetA2 inhibitedthe angiogenesis and growth of the tumors in comparison to untreatedcontrol.

Animal Tumors.

Oral administration of 60 mg/kg/day SHetA2 inhibited the development ofblood vessels inside melanoma, ovarian and renal cancer tumorsestablished in mice. FIG. 12 demonstrates SHetA2 administration reducedstaining of the anti-CD34 antibody, which specifically binds theendothelial cells that line blood vessels, in treated tumors incomparison to normal. SHetA2 improved survival in the melanoma andovarian cancer models. Survival was the primary endpoint of thosestudies. SHetA2 decreased tumor growth in the renal cancer model. Tumorgrowth was the primary endpoint of the renal cancer study.

Validation of Thymidine Phosphorylase (TP) and Thrombospondin 4 (TSP4)Inhibition.

The ability of SHetA2 to inhibit expression of TP and TSP-4 at the RNAand protein levels were validated using real time polymerase chainreaction and Western blot analysis as shown in FIGS. 13 and 14.

SHetA2 Inhibition of NF-κB.

Inhibition of NF-κB activity inhibits angiogenesis by suppressingexpression of vascular endothelial growth factor (VEGF) andinterleukin-8 (IL-8) in an animal model of ovarian cancer. Because bothTP and TSP4 have DNA binding sites for the NFκB transcription factor intheir promoter, the ability of SHetA2 to inhibit activity of NF-κB inovarian cancer cultures was tested using a luciferase reporter plasmiddriven by NF-κB sites in the promoter. Rapid inhibition of activity wasobserved within 30 minutes suggesting a relationship to themitochondrial mechanism of SHetA2. Inhibition increased over the 4 hourevaluation time (FIG. 15).

In Vivo Inhibition of Xenograft Tumor Growth.

The ability of retinoids to inhibit the growth of ovarian carcinomas invivo was evaluated in a nude mouse xenograft model using the humanOVCAR-3 ovarian carcinoma cell line. All treatments, 4-HPR, SHet50 andSHetA2, significantly inhibited the growth of the xenograft tumors (FIG.4). After 15 days of treatment, the relative volumes of the tumors inthe retinoid treated groups were significantly smaller than the controlgroup treated with sesame oil alone (t-test on day 29: p=0.028 for4-HPR; p=0.010 for SHet50; p=0.046 for SHetA2). There were nosignificant differences between the degrees of growth inhibition exertedby the different drugs.

Regulation of Angiogenic Gene Expression.

Microarray analysis demonstrated that SHetA2-treated ovarian cancercells exhibit significantly altered expression of specific genes (seeTable 5).

TABLE 5 Gene Name Genbank ID Fold Disintegrin-like and metalloprotease(reprolysin type) with NM_007038.1 0.23 thrombospondin type 1 motif(ADAMTS5) Disintegrin-like and metalloprotease (reprolysin type) withAK023795.1 0.43 thrombospondin type 1 motif, highly similar to Homosapiens metalloproteinase with thrombospondin type 1 motifs (ADAMTS1)Vascular endothelial growth factor (VEGF) AF022375.1 0.47 Figroblastgrowth factor 2 (basic)(FGF2 NM_002006.1 0.35 Thrombospondin 1 (THBS1)BE999967 0.42 Fibroblast growth factor 18 (FGF18) NM_003862.1 2.75Thromospondin 4 (THBS4) NM_03248.1 2.58 Angiopoietin-like 2 (ANGPTL2)NM_012098.1 2.16 Glypican-6 (GPC6) AF111178.1 2.07 METH1 protein(METH1), contain a repeated amino acid motif AF060152.1 0.39 homologousto the anti-angiogenic type 1 repeats of thromospondin-1 (TSP1)Thrombospondin 4 NM_003248 0.58 Endothelial cell growth factor 1(platelet-derived)/Thymidine NM_001953 0.42 phosphorylase

Effects of Clinically Relevant Concentrations.

Although micromolar concentrations of retinoids have been achieved inphase I clinical trials, lower effective doses are desirable. Ademonstration that less than micromolar concentrations of a retinoidcould exhibit chemopreventative activity was demonstrated for 4-HPR,which was evaluated in a randomized clinical trial conducted at theIstituto Nazionale Tumori in Italy. In this trial, women who had surgeryfor breast cancer were randomly assigned to receive 200 mg of 4-HPR perday or a placebo for five years to determine whether 4-HPR reduced theincidence of second primary tumors. A secondary finding of this trialwas that statistically significantly fewer ovarian cancers developed inwomen who took 4-HPR. 4-HPR is similar to classical retinoids in that itboth can induce differentiation in a receptor-dependent manner at 1 μM.At 3 to 12.5 μM however, 4-HPR differs from classical retinoids in thatit induces apoptosis in a variety of cell lines through areceptor-independent mechanism. In the Italian trial, the plasmaconcentration of 4-HPR achieved in treated women ranged from 340 to 868μM. Therefore, the concentrations in the ovarian tissue of treated womenwere not likely to have reached the levels required for apoptosis. Wehypothesized that the 4-HPR chemoprevention observed was due toinduction of differentiation or apoptosis in ovarian cancer tissue bychronic exposure to less than micromolar 4-HPR concentrations.

To test our hypotheses, we evaluated the effects of clinicallyachievable concentrations of 4-HPR and Flex-Hets on growth,differentiation and apoptosis in ovarian tissue. A series of compoundswith a variety of receptor activation profiles were evaluated. In orderto evaluate therapeutic effects, we developed an organotypic culturemodel of ovarian tumors by growing established cell lines and primarycultures inside collagen gels. Monolayer cultures of cells are notaccurate representations of in vivo tissue because they lack the typesof interactions that cells have with other cells and with theextracellular matrix in vivo. These interactions are known to influencegene expression and thus the constitution and behavior of cells. In ourorganotypic model, the growth of tumor cells inside the collagen allowsa representation of tumors that have invaded stromal tissue. In thepresence of fibroblasts, established cell lines and primary cultures ofovarian cancer formed colonies inside the collagen, while normal orbenign ovarian epithelial cells remained as single cells inside thecollagen. Human ovarian cancer cell lines (OVCAR-3, Caov-3, SK-OV-3,A2780, UCI 101) and primary human cultures (borderline ovarian tumor(O1), serous papillary tumor (O2), benign ovarian cyst (O3)) werecultured inside and on top of a collagen gel seeded with 3T3 fibroblastsfor one week. After further incubation in the presence or absence of 1μM retinoid (media concentration) for 3, 7 or 14 days, cultures werefixed and sectioned for immunohistochemical analysis. Eleven differentretinoids were evaluated and SHetA2 was found to be the most potentregulator of growth, differentiation and apoptosis as published andsummarized below.

Induction of Apoptosis.

Two different assays (Annexin-V-Flous and TUNEL) performed in severalcell lines demonstrated that cell loss in SHetA2-treated cultures wasdue to induction of apoptosis. The Annexin-V-Flous assay, demonstratedthat SHetA2 induced apoptosis cancer cell lines in the sameconcentration range as 4-HPR (FIG. 16). The TUNEL assay was used toevaluate apoptosis in fixed, paraffinembedded, cut sections of thetreated and control cultures (FIG. 17). Apoptosis was induced by 4-HPRand all of the 3-atom linker receptor-independent FHs, SHetA2, SHetA3and SHetA4, and by two potent receptor pan agonist retinoids, 9-cis-RAand SHet50. The 2-atom ester linker Flex-Hets exhibiting a variety ofreceptor specificities did not induce apoptosis. SHetA2 induced thehighest level of apoptosis. Extensive review of multiple sections andrepetitions of this experiment did not reveal any cellular clumps in theorganotypic cultures that were undergoing simultaneous differentiationand apoptosis. These two cellular outcomes appeared to be mutuallyexclusive. These results demonstrate that SHetA2 specifically inducesapoptosis and is not just cytotoxic.

Demonstration of the Role of Thymidine Phosphorylase in SHetA2Apoptosis.

Ovarian cancer cells were treated with SHetA2 in the presence andabsence of Thymidine Phosphorylase, which inhibited the ability ofSHetA2 to induce apoptosis and kill the cells (FIG. 18).

Chemoprevention Activity of SHetA2.

FIG. 19 illustrates a typical experiment in which organotypic culturesof normal endometrial cells were treated with a carcinogen called DMBAand developed the cancer phenotype. Treatment with SHetA2 prevented thedevelopment of this phenotype and induced apoptosis in cells thatsurvived. The cultures were made by growing normal cells inside a3-dimensional collagen matrix for two weeks to allow tissue to develop.Transformation of the normal cells to cancer cells as observed byhistologic examination was confirmed by karyometric analysis of thenuclear abnormalities as can be see in the bar graphs to the right ofthe photomicrographs. Karyometric analysis is a statistical analysis ofthe spatial distribution of the chromatin pattern that acts as anintegrating biomarker. Images were acquired using a 63× high NA oilimmersion objective, converted to optical density and stored digitally,after which nuclei were segmented from the general image using customsoftware. One hundred nuclei were randomly selected from each sample foranalysis. Only well defined nuclei free of debris or overlap with othernuclei were chosen. All segmented nuclei were then subjected to texturefeature extraction using TICAS software, resulting in 95 texturefeatures for each nucleus that were subjected to quantitative analysis.Karyometric analysis also demonstrated that SHetA2 prevents thiscarcinogenic transformation.

Treatment of Polycystic Kidney Disease

We have found that flexible heteroarotinoids inhibit cyst formation in a3-dimensional gel culture system using human polycystic kidney diseasecells. Thus, the invention is also directed to a novel use of flexibleheteroarotinoids as an in vivo treatment for polycystic kidney diseaseto reduce, inhibit or prevent cyst formation.

The 3-D model of the PKD was developed as described here. Single-cellsuspensions of the PCKD cell lines was re-suspended in a 4□C solutionmixture containing rat tail collagen I (Collaborative BiomedicalProducts, Bedford, Mass.), matrigel (Becton Dickinson) and MEM. Thesuspension was poured into Falcon cell culture inserts with transparentmembranes containing 0.4 μm pores (Becton Dickinson, Franklin Lakes,N.J.) and placed at 37□C. Treatment of cultures was initiated after 1day of growth and replenished with medium once in every two days. Drugs(Flex-Hets at 4 μM Conc.) and hormones (PGE2 at 25 ng/ml) wereadministered into the medium surrounding the insert. Untreated controlcultures were grown in the same conditions as that of the treatedcultures. Drug treatment was carried out for 3 weeks before theireffects on growth and inhibition were evaluated.

The inhibitory and differentiation ability of two different Flex-NetsShetA3 and SHetA4 was evaluated in the 3-D model. Cysts were observed inboth control as well as PGE2 treated 3-D cultures, with the laterinducing several cysts which were also very huge in size. In culturestreated with Flex-Hets, a 4 μM concentration of these drugs completelyeliminated cyst formation (FIG. 21). Interestingly, both Flex-Hetsinduced the formation of several branched tubular structures that ischaracteristic of a differentiated phenotype in kidney tissue. Theseresults indicate that Flex-Hets can be used to potently inhibit cystformation in PKD.

Treatment of Disorders of Glycoprotein Metabolism and Lysosomal StorageDiseases.

In another embodiment of the invention, SHetA2 regulates expression ofgenes involved in glycoprotein metabolism and related lysosomal storagediseases (LSDs) and galactosemia. SHetA2 regulates expression of bothHomo sapiens galactosamine (N-acetyl)-6-sulfate sulfatase (GALNS), whichis commonly mutated in Morquios syndrome, mucopolysaccharidosis typeIVA, which is a lysozomal storage disease and Homo sapiensUDP-galactose-4-epimerase (GALE), which causes galactosemia. The resultof this regulation is altered patterns of glycoprotein staining intumors. The ability of SHetA2 to regulate the pattern of glycoproteinsin vivo was tested by staining sections of human xenograft tumors fromSHetA2-treated mice and corresponding untreated control animals. SHetA2was shown to increase staining for mucin-1 (MUC1) protein and to revertthe abnormal pattern of MUC1 staining on all cell surfaces in untreatedovarian tumors to the normal pattern of luminal only staining in thetreated tumors (FIG. 5). In addition, periodic acid schiff (PAS) stainof renal xenograft tumors demonstrated an increase in generalglycoprotein expression and lysosomal staining in the treated tumors incomparison to the untreated tumors (FIG. 20).

LSDs are a group of more than 40 genetic disorders caused by inbornerrors of metabolism (which are problems in the genes that affect howcells break down certain molecules). People with LSDs are either lackingor in short supply of particular enzymes that are found in lysosomes.Because of this, molecules that are meant to be broken down by themissing enzymes build up within the lysosomes, and can prevent the cellfrom working properly. Separately, lysosomal storage diseases are eachrare diseases. As a group, lysosomal storage diseases are estimated toaffect 1 in 7,700 live births.

Lysosomal storage diseases which are contemplated for treatment by theFlex-Hets described herein include, but are not limited to, Battendisease, Fabry disease, Gaucher disease, Krabbe disease,Mucopoly-sacchiradosis I (MPS I/Hurler/Hurler-Scheie/Scheie),Mucopolysacchiradosis II (MPS II/Hunter Disease), Niemann-Pick disease,Pompe disease, and Tay-Sachs disease.

In summary, and without wishing to be constrained by theory, it isthought that various mechanisms of action for the treatment of thesediseases and conditions by the flexible heteroarotinoids include, butare not limited to: (1) reversal of abnormal differentiation in diseasestates such as cancer, (2) normalization of mutant HNF-4 function totreat diabetes, (3) normalization of Factor IX levels by HNF-4regulation to treat hemophelia, (4) restoration of HNF-4 levels and/orfunction to treat liver failure, (5) normalization of human aldehydedehydrogenase 2 expression, (6) normalization of cholesterol levelsthrough regulation of expression of genes involved in cholesterolmetabolism, (7) treatment of obesity through regulation of expression ofgenes involved in lipid metabolism, (8) treatment of high triglyceridesthrough regulation of expression of genes involved in triglyceridemetabolism, (9) treatment of disorders involving glycoprotein metabolismand lysosomal storage diseases, and (10) treatment of metabolicdisorders through regulation of expression of genes involved inmetabolism.

Microarray Analysis

Microarray analysis was conducted to determine specific disease causinggenes which were expressed at higher (>1 fold) or lower (<1 fold) levelsthan normal in ovarian cancer cells treated with SHetA2 (see Table 6).

TABLE 6 Gene Name Genbank ID Fold Eye Diseases Vitelliform maculardystrophy (Best disease, bestrophin) (VMD2) NM_004183.1 0.28 Similar tovitelliform macular dystrophy (Best disease, bestrophin) BC041664.1 0.26Retinitis pigmentosa GTPase regulator (RPGR) NM_000328.1 0.47 Keratocan(KERA) involved in cornea plana NM_007035.2 0.50 Neuromuscular DiseasesAmyotrophic lateral sclerosis 2 (juvenile) chromosome region,NM_015049.1 0.41 candidate 3 (ALS2CR3) ESTs, Weakly similar toDRPL_Human Atrophin-1 (DRPL) in AI358954 2.52dentatorubral-pallidoluysian atrophy ESTs, Weakly similar to DRPL_HumanAtrophin-1 (DRPL) in AI358954 2.52 Huntington's disease (HD) anddentatorubral and pallidoluysian atrophy (DRPLA)dentatorubral-pallidoluysian atrophy ATP-binding cassette, sub-family D(ALD), member 2 (ABCD2) in X- NM_005164.1 2.71 linkedadrenoleukodystrophy Peroxisomal biogenesis factor 14 (PEX14) inperoxisome NM_004565.1 2.06 biogenesis disorders SNRPN upstream readingframe (SNURF) in Prader-Willi and BF114870 0.39 Angelman syndrome Smcxhomolog, X chromosome (mouse) in nonsyndromic X-linked NM_004187 0.61mental retardation Autoimmune Diseases Butyrophilin, subfamily 3, memberA2 (BTN3A2) milk protein AI991252 2.31 Butyrophilin (BTF3) - Milk FatCluster Incl. 2.15 U90548 Butyrophilin, subfamily 3, member A3 (BTN3A3)NM_006994.2 2.04 Familial Mediterranean fever locus region AF098968.10.30 Regulatory factor X-associated protein (RFXAP) in bare lymphocyteNM_000538.1 0.41 syndrome Peroxisomal biogenesis factor 3 (PEX3)NM_003630.1 0.43 Skeletal Diseases Trichorhinophalangeal syndrome I gene(TRPS1) NM_014112.1 0.36 Dystrophin (muscular dystrophy, Duchenne andBecker types), NM_004019.1 2.76 dystrophin Dp40 isoform (DMD)Facioscapulohumeral muscular dystrophy (FSHD) gene region, D4Z4 D380242.43 tandem repeat unit Dysferlin, limb girdle muscular dystrophy 2B(autosomal recessive) NM_003494.1 2.16 (DYSF) Sarcoglycan, gamma (35 kDdystrophin-associated glycoprotein) NM_000231.1 0.04 (MuscularDystrophy) Storage and Metabolism Deficiencies Hemochromatosis splicevariant 495-2314del, Iron Storage Disorder AF144243.1 3.01 Nephropontininhibits urine crystals - kidney stones M83248.1 2.34 Galactosamine(N-acetyl)-6-sulfate sulfatase in Morquio syndrome, NM_000512 0.52mucopolysaccharidosis type IVA UDP-galactose-4-epimerase inepimerase-deficiency galactosemia NM_000403 0.66 Polycystic KidneyDisease Prothymosin alpha (PTMA) AF257099 2.60 Cancer Prostate cancerassociated protein 5 (PCANAP5) AI299378 0.23 FYN oncogene related toSRC, FGR and YES (FYN) N20923 0.13 Myeloidlymphoid leukemia 2 (MLL2)AF105279.1 0.31 Deleted in lymphocytic leukemia, 2/DEF = Homo sapiensBCMS- AF264787.1 0.33 upstream neighbor (BCMSUN) Neuroblastoma,suppression of tumorigenicity 1 (NBL1), NM_005380.1 3.07 v-Myc avianmyelocytomatosis viral related oncogene, BC002712.1 3.99 neuroblastomaderived (MYCN) Myeloidlymphoid or mixed-lineage leukemia (trithorax(Drosophila) AB016902.2 5.07 homolog); translocated to, 4 (HGC6.3) ESTs,Moderately similar to S72399 ras-homolog GTPase rab28 AI080106 7.75isoform S FYN oncogene related to SRC, FGR and YES, c-syn protooncogeneM14333.1 0.44 Novel MAFF (v-maf musculoaponeurotic fibrosarcoma (avian)Cluster Incl. 0.47 oncogene family, protein F) LIKE protein AL021977Nasopharyngeal carcinoma susceptibility protein (LZ16) NM_013275.1 0.41RNA binding motif protein 5, putative tumor suppressor (LUCA15) U23946.10.43 Promyelocytic leukemia (PML) NM_002675.1 2.76 RASSF3 isoform A,putative tumor suppressor AY062002.1 2.88 Chromosome 5 open readingframe 4 (C5ORF4), putative tumor NM_016348.1 2.76 suppressor B-cellCLLlymphoma 3 (BCL3) NM_005178.1 2.05 B-cell CLLlymphoma 3 (BCL3)AI829875 2.58 Cervical cancer suppressor gene-4 protein (HCCS-4)AF465843.1 2.06 Deleted in lymphocytic leukemia, BCMS-upstream neighborAF264787.1 0.40 (BCMSUN) (DLEU2) Niban protein (NIBAN) (Renalcarcinogenesis) NM_022083.1 0.21 BAGE2 antigen (BAGE2) AF218570.1 0.34BAGE2 antigen (BAGE2) AF218570.1 0.34 NICE-5 protein AW779859 0.48Unknown protein mRNA within the p53 intron 1 U58658.1 0.45 Putativec-Myc-responsive (RCL) AA523444 0.41 Enhancer of zeste (Drosophila)homolog 2 (EZH2) (USP14) NM_004456.1 0.47 EHT protein AF068266.1 2.80BAM in multiple myeloma AF455755.1 3.05 Lipoma cell line Li-538SV40ectopic sequence from HMGI-C fusion AI990940 0.47 mRNA, 3 sequence (LI)ASPLTFE3 type 2 fusion protein, ASPLTFE3 type 1 fusion proteinAY034077.1 0.46 mRNA in Sarcoma PNAS-128 in leukemia AF277186.1 0.36Rhabdoid tumor deletion region protein 1 (RTDR1) NM_014433.1 2.20 IMAGE:3050107 mRNA, TBC1 domain family BC001525 0.67 FLJ23538 fis, highlysimilar to BETA2 Human MEN1 (Multiple AI042152 0.31 Endocrine Neoplasia)region clone epsilonbeta (BETA2) N-cym protein NM_006316 2.38Transglutaminase 4 (prostate) NM_003241 0.24 Flex-Het Regulated GenesKnown to Cause Specific Diseases. Genes that were expressed atstatistically significant higher (>1 Fold) or lower (<1 Fold) levels inovarian cancer cells treated with Flex-Het 11 (SHetA2) were identifiedby microarray analysis.

Utility

In summary, among the diseases or conditions which can benefit fromtreatment with flexible heteroarotinoids as described herein are, (1)cancers and other diseases that involve abnormal differentiation, (2)diabetes, (3) hemophelia, (4) liver disease, (5) diseases involvinghuman aldehyde dehydrogenase 2, (6) polycystic kidney disease, (7)lysosomal storage diseases, (8) high cholesterol, (9) obesity, (10) hightriglycerides, (11) diseases of glycoprotein metabolism, (12) diseasesinvolving abnormal angiogenesis, and (13) diseases caused or enhanced bygenes shown in Table 5 and Table 6.

The present invention provides a method for the treatment of a patientafflicted with such diseases by administration of a therapeuticallyeffective amount of a flexible heteroarotinoid.

As defined herein, a therapeutically effective amount of a Flex-Het ofthe present invention refers to an amount which is effective incontrolling, reducing, or inhibiting a disease or condition as describedherein. The term “controlling” is intended to refer to all processeswherein there may be a slowing, interrupting, arresting, or stopping ofthe progression of the disease and does not necessarily indicate a totalelimination of all disease symptoms.

The term “therapeutically effective amount” is further meant to definean amount of an Flex-Het resulting in the improvement of any parametersor clinical symptoms characteristic of a disease or condition describedherein. The actual dose will be different for the various specificFlex-Hets, and will vary with the patient's overall condition, theseriousness of the symptoms, and counter indications.

As used herein, the term “subject” or “patient” refers to a warm bloodedanimal such as a mammal which is afflicted with a particularinflammatory disease state. It is understood that guinea pigs, dogs,cats, rats, mice, livestock, horses, cattle, sheep, zoo animals, andhumans are examples of animals within the scope of the meaning of theterm.

A therapeutically effective amount of the Flex-Het used in the treatmentdescribed herein can be readily determined by the attendingdiagnostician, as one skilled in the art, by the use of conventionaltechniques and by observing results obtained under analogouscircumstances. In determining the therapeutically effective dose, anumber of factors are considered by the attending diagnostician,including, but not limited to: the species of mammal; its size, age, andgeneral health; the specific disease or condition involved; the degreeof or involvement or the severity of the disease or condition; theresponse of the individual subject; the particular compoundadministered; the mode of administration; the bioavailabilitycharacteristic of the preparation administered; the dose regimenselected; the use of concomitant medication; and other relevantcircumstances.

A therapeutically effective amount of the compositions of the presentinvention will generally contain from about 0.01 μg/kg to about 100mg/kg (weight of active ingredient/body weight of patient) of aFlex-Het. Preferably, the composition will deliver at least 0.1 μg/kg to50 mg/kg, and more preferably at least 1 μg/kg to 10 mg/kg of aFlex-Het.

Practice of the method of the present invention comprises administeringto a subject a therapeutically effective amount of the Flex-Het, in anysuitable systemic or local formulation, in an amount effective todeliver the dosages listed above. For example, an effective,particularly preferred dosage of the Flex-Het for example for inhibitingangiogenesis, ovarian cancer or PKD is 1 μg/kg to 1 mg/kg of theFlex-Het. The dosage can be administered on a one-time basis, or (forexample) from one to five times per day or once or twice per week, orcontinuously via a venous drip, depending on the desired therapeuticeffect. Flex-Hets can be used in preparations that contain additionalpharmaceuticals for combination therapies, and can be used incombination with other pharmaceuticals administered separately.

As noted, preferred amounts and modes of administration are able to bedetermined by one skilled in the art. One skilled in the art ofpreparing formulations can readily select the proper form and mode ofadministration depending upon the particular characteristics of theFlex-Het selected, the disease state to be treated, the stage of thedisease, and other relevant circumstances using formulation technologyknown in the art, described, for example, in Remington's PharmaceuticalSciences, latest edition, Mack Publishing Co.

Pharmaceutical compositions can be manufactured utilizing techniquesknown in the art. Typically the therapeutically effective amount of theFlex-Het will be admixed with a pharmaceutically acceptable carrier.

The Flex-Het compositions of the present invention may be administeredby a variety of routes, for example, topically, orally or parenterally(i.e., subcutaneously, intravenously, intramuscularly,intraperitoneally, or intratracheally).

For oral administration, the Flex-Nets can be formulated into solid orliquid preparations such as capsules, pills, tablets, lozenges, melts,powders, suspensions, or emulsions. Solid unit dosage forms can becapsules of the ordinary gelatin type containing, for example,surfactants, lubricants and inert fillers such as lactose, sucrose, andcornstarch or they can be sustained release preparations.

In another embodiment, the Flex-Hets of this invention can be tablettedwith conventional tablet bases such as lactose, sucrose, and cornstarchin combination with binders, such as acacia, cornstarch, or gelatin,disintegrating agents such as potato starch or alginic acid, and alubricant such as stearic acid or magnesium stearate. Liquidpreparations are prepared by dissolving the active ingredient in anaqueous or non-aqueous pharmaceutically acceptable solvent which mayalso contain suspending agents, sweetening agents, flavoring agents, andpreservative agents as are known in the art.

For parenteral administration, the Flex-Nets may be dissolved in aphysiologically acceptable pharmaceutical carrier and administered aseither a solution or a suspension. Illustrative of suitablepharmaceutical carriers are water, saline, dextrose solutions, fructosesolutions, ethanol, or oils of animal, vegetative, or synthetic origin.The pharmaceutical carrier may also contain preservatives, and buffersas are known in the art.

The Flex-Hets of this invention can also be administered topically. Thiscan be accomplished by simply preparing a solution of the compound to beadministered, preferably using a solvent known to promote transdermalabsorption such as ethanol or dimethyl sulfoxide (DMSO) with or withoutother excipients. Preferably topical administration will be accomplishedusing a patch either of the reservoir and porous membrane type or of asolid matrix variety.

As noted above, the compositions can also include an appropriatecarrier. For topical use, any of the conventional excipients may beadded to formulate the active ingredients into a lotion, ointment,powder, cream, spray, or aerosol. For surgical implantation, the activeingredients may be combined with any of the well-known biodegradable andbioerodible carriers, such as polylactic acid and collagen formulations.Such materials may be in the form of solid implants, sutures, sponges,wound dressings, and the like. In any event, for local use of thematerials, the active ingredients usually be present in the carrier orexcipient in a weight ratio of from about 1:1000 to 1:20,000, but arenot limited to ratios within this range. Preparation of compositions forlocal use are detailed in Remington's Pharmaceutical Sciences, latestedition, (Mack Publishing).

Additional pharmaceutical methods may be employed to control theduration of action of the Flex-Hets. Increased half-life and controlledrelease preparations may be achieved through the use of polymers toconjugate, complex with, or absorb the Flex-Nets described herein. Thecontrolled delivery and/or increased half-life may be achieved byselecting appropriate macromolecules (for example, polysaccharides,polyesters, polyamino acids, homopolymers polyvinyl pyrrolidone,ethylenevinylacetate, methylcellulose, or carboxymethylcellulose, andacrylamides such as N-(2-hydroxypropyl) methacrylamide, and theappropriate concentration of macromolecules as well as the methods ofincorporation, in order to control release.

Another possible method useful in controlling the duration of action bycontrolled release preparations and half-life is incorporation of theFlex-Het molecule or its functional derivatives into particles of apolymeric material such as polyesters, polyamides, polyamino acids,hydrogels, poly(lactic acid), ethylene vinylacetate copolymers,copolymer micelles of, for example, PEG and poly(1-aspartamide).

The half-life of the Flex-Nets described herein can be extended by theirbeing conjugated to other molecules such as polymers using methods knownto persons of ordinary skill in the art to form drug-polymer conjugates.For example, the Flex-Hets can be bound to molecules of inert polymersknown in the art, such as a molecule of polyethylene glycol (PEG) in amethod known as “pegylation”. Pegylation can therefore extend the invivo lifetime and thus therapeutic effectiveness of the Flex-Hetmolecule. Pegylation also reduces the potential antigenicity of theFlex-Het molecule. Pegylation can also enhance the solubility ofFlex-Hets thereby improving their therapeutic effect. PEGs used may belinear or branched-chain.

By “pegylated Flex-Het” is meant a Flex-Het of the present inventionhaving a polyethylene glycol (PEG) moiety covalently bound to a linkinggroup of the Flex-Het.

By “polyethylene glycol” or “PEG” is meant a polyalkylene glycolcompound or a derivative thereof, with or without coupling agents orderivatization with coupling or activating moeities (e.g., with thiol,triflate, tresylate, aziridine, oxirane, or preferably with a maleimidemoiety). Compounds such as maleimido monomethoxy PEG are exemplary oractivated PEG compounds of the invention. Other polyalkylene glycolcompounds, such as polypropylene glycol, may be used in the presentinvention. Other appropriate polymer conjugates include, but are notlimited to, non-polypeptide polymers, charged or neutral polymers of thefollowing types: dextran, colominic acids or other carbohydrate basedpolymers, biotin deriviatives and dendrimers, for example. The term PEGis also meant to include other polymers of the class polyalkyleneoxides.

The chemically modified Flex-Nets contain at least one PEG moiety,preferably at least two PEG moieties, up to a maximum number of PEGmoieties bound to the Flex-Het without abolishing activity.

The PEG moiety attached to the Flex-Het may range in molecular weightfrom about 200 to 20,000 MW. Preferably the PEG moiety will be fromabout 1,000 to 8,000 MW, more preferably from about 3,250 to 5,000 MW,most preferably about 5,000 MW.

The actual number of PEG molecules covalently bound per chemicallymodified Flex-Het of the invention may vary widely depending upon thedesired Flex-Het stability (i.e. serum half-life).

Alternatively, it is possible to entrap the Flex-Het in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization (for example, hydroxymethylcellulose orgelatine-microcapsules and poly-(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules), or in macroemulsions. Such techniques are disclosed inthe latest edition of Remington's Pharmaceutical Sciences.

U.S. Pat. No. 4,789,734 describes methods for encapsulating biochemicalsin liposomes and is hereby expressly incorporated by reference herein.Essentially, the Flex-Het is dissolved in an aqueous solution, theappropriate phospholipids and lipids added, along with surfactants ifrequired, and the material dialyzed or sonicated, as necessary. A reviewof known methods is by G. Gregoriadis, Chapter 14. “Liposomes”, DrugCarriers in Biology and Medicine, pp. 287-341 (Academic Press, 1979).Microspheres formed of polymers or proteins are well known to thoseskilled in the art, and can be tailored for passage through thegastrointestinal tract directly into the blood stream. Alternatively,the agents can be incorporated and the microspheres, or composite ofmicrospheres, implanted for slow release over a period of time, rangingfrom days to months. See, for example, U.S. Pat. Nos. 4,906,474;4,925,673; and 3,625,214 which are incorporated by reference herein.

When the composition is to be used as an injectable material, it can beformulated into a conventional injectable carrier. Suitable carriersinclude biocompatible and pharmaceutically acceptable phosphate bufferedsaline solutions, which are preferably isotonic.

For reconstitution of a lyophilized product in accordance with thisinvention, one may employ a sterile diluent, which may contain materialsgenerally recognized for approximating physiological conditions and/oras required by governmental regulation. In this respect, the sterilediluent may contain a buffering agent to obtain a physiologicallyacceptable pH, such as sodium chloride, saline, phosphate-bufferedsaline, and/or other substances which are physiologically acceptableand/or safe for use. In general, the material for intravenous injectionin humans should conform to regulations established by the Food and DrugAdministration, which are available to those in the field.

The pharmaceutical composition may also be in the form of an aqueoussolution containing many of the same substances as described above forthe reconstitution of a lyophilized product.

The compounds can also be administered as a pharmaceutically acceptableacid- or base-addition salt, formed by reaction with inorganic acidssuch as hydrochloric acid, hydrobromic acid, perchloric acid, nitricacid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organicacids such as formic acid, acetic acid, propionic acid, glycolic acid,lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,maleic acid, and fumaric acid, or by reaction with an inorganic basesuch as sodium hydroxide, ammonium hydroxide, potassium hydroxide, andorganic bases such as mono-, di-, trialkyl and aryl amines andsubstituted ethanolamines.

As mentioned above, the Flex-Nets of the invention may be incorporatedinto pharmaceutical preparations which may be used for therapeuticpurposes. However, the term “pharmaceutical preparation” is intended ina broader sense herein to include preparations containing a Flex-Hetcomposition in accordance with this invention, used not only fortherapeutic purposes but also for reagent or diagnostic purposes asknown in the art, or for tissue culture. The pharmaceutical preparationintended for therapeutic use should contain a “pharmaceuticallyacceptable” or “therapeutically effective amount” of a Flex-Het, i.e.,that amount necessary for preventative or curative health measures. Ifthe pharmaceutical preparation is to be employed as a reagent ordiagnostic, then it should contain reagent or diagnostic amounts of aFlex-Het.

All of the assay methods listed herein are well within the ability ofone of ordinary skill in the art given the teachings provided herein.

All references, patents and patent applications cited herein are herebyexpressly incorporated herein in their entireties by reference.

Changes may be made in the various methods and compositions describedherein without departing from the spirit and scope of the invention asdefined in the following claims.

CITED REFERENCES

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1. A method for treating or inhibiting polycystic kidney disease in asubject in need of treatment thereof or at risk therefor, comprising:providing a quantity of a flexible heteroarotinoid having a urea orthiourea linker; and administering the flexible heteroarotinoid to thesubject.
 2. The method of claim 1 wherein the subject is a mammal. 3.The method of claim 1 wherein the subject is a human.
 4. The method ofclaim 1 wherein the flexible heteroarotinoid is selected from the groupconsisting of SHetA2, SHetA3, and SHetA4.
 5. The method of claim 1wherein the flexible heteroarotinoid is provided in a compositioncomprising a pharmaceutically-acceptable carrier.
 6. A method fortreating or inhibiting a lysosomal storage disease in a subject in needof such treatment, comprising: providing a quantity of a flexibleheteroarotinoid having a urea or thiourea linker; and administering theflexible heteroarotinoid to the subject.
 7. The method of claim 6wherein the lysosomal storage disease is selected from the groupconsisting of Batten disease, Fabry disease, Gaucher disease, Krabbedisease, Mucopolysacchiradosis I (MPS I/Hurler/Hurler-Scheie/Scheie),Mucopolysacchiradosis II (MPS II/Hunter Disease), Niemann-Pick disease,Pompe disease, and Tay-Sachs disease.
 8. The method of claim 6 whereinthe subject is a mammal.
 9. The method of claim 6 wherein the subject isa human.
 10. The method of claim 6 wherein the flexible heteroarotinoidis selected from the group consisting of SHetA2, SHetA3, and SHetA4. 11.The method of claim 6 wherein the flexible heteroarotinoid is providedin a composition comprising a pharmaceutically-acceptable carrier.